LOCUS       sequence-181522-        6080 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E.coli version of EPN01(∆M).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6080)
  AUTHORS   Votteler J, Ogohara C, Yi S, Hsia Y, Nattermann U, Belnap DM, King 
            NP, Sundquist WI
  TITLE     Designed proteins induce the formation of nanocage-containing 
            extracellular vesicles.
  JOURNAL   Nature. 2016 Dec 8;540(7632):292-295. doi: 10.1038/nature20607. Epub
            2016 Nov 30.
  PUBMED    27919066
REFERENCE   2  (bases 1 to 6080)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/nature20607"; journalName: "Nature"; date: "2016-12-8- 8"; 
            volume: "540"; issue: "7632"; pages: "292-295"
COMMENT     Sequence Label: pET29b-EPN01(∆M)-His
FEATURES             Location/Qualifiers
     source          1..6080
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     149..168
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     256..275
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        256..274
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    275..299
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             314..336
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1007..1036
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1196..1213
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1266..1284)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1280..1327
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1364..1819
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1451..1470)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1661..1682
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1911..2726)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1911..2726)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2634..2653
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2718..2737
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2848..3436
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3337..3356
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3590..3607
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3622..3761
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3687..3706)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3847..3869
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3863..4054)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4863..5945)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5907..5926
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5946..6023)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca
       61 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
      121 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
      181 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatcgatc
      241 tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc
      301 ctctagaaat aattttgttt aactttaaga aggagatata catatggctg cgcgtgcgtc
      361 cggttctaaa tctggctccg gctctgactc tggtagcaag atcgaagagc tgttcaagaa
      421 acacaagatc gttgccgtgc tgcgtgccaa tagtgtggaa gaagcgaaaa agaaagcgct
      481 ggcggttttc ctgggcggcg ttcatctgat tgaaattacc tttaccgtgc cggatgcgga
      541 taccgtgatt aaggaactga gctttctgaa ggaaatgggc gcgattattg gtgcgggcac
      601 cgtgaccagc gtggagcagt gccgtaaagc ggtggaaagt ggcgccgaat tcattgtgag
      661 tccgcacctg gacgaggaaa ttagccaatt ttgcaaggag aagggtgtgt tctatatgcc
      721 aggcgttatg accccgaccg aactggtgaa agccatgaaa ctgggccata ccatcttaaa
      781 actgtttccg ggtgaggtgg tgggtccgca gtttgttaaa gcgatgaaag gtccgtttcc
      841 gaatgtgaaa tttgtgccaa ccggcggtgt taatctggac aatgtgtgcg aatggttcaa
      901 agcgggcgtg ctggccgtgg gcgtgggcag cgcgttagtg aaaggcaccc cggtggaagt
      961 ggcggaaaag gccaaggcgt tcgttgagaa gattcgtggc tgcaccgaac agaaactgat
     1021 tagcgaagaa gatctgcagt ctcgtccgga accgaccgca ccgccggaag aatctttccg
     1081 ttctggtgtt gaaaccacca cgccaccgca aaaacaggag ccgatcgaca aagaactgta
     1141 cccgctgacc tctctgcgtt ctctgttcgg taacgacccg tcttctcagc tcgagcacca
     1201 ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     1261 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     1321 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc
     1381 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     1441 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     1501 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     1561 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     1621 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     1681 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     1741 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     1801 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     1861 gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac
     1921 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     1981 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     2041 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     2101 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     2161 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc
     2221 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     2281 agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
     2341 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     2401 acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta
     2461 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
     2521 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     2581 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
     2641 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa
     2701 gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
     2761 agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     2821 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     2881 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     2941 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     3001 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     3061 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     3121 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     3181 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     3241 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     3301 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     3361 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     3421 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3481 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     3541 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     3601 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3661 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     3721 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     3781 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     3841 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     3901 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     3961 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     4021 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     4081 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     4141 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     4201 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     4261 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     4321 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     4381 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     4441 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     4501 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga
     4561 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     4621 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     4681 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     4741 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     4801 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     4861 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4921 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     4981 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     5041 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     5101 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatgtccgc accaacgcgc
     5161 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     5221 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     5281 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     5341 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     5401 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     5461 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     5521 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     5581 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     5641 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     5701 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     5761 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     5821 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     5881 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     5941 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     6001 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     6061 taggaagcag cccagtagta
//
